2974 • The Journal of Neuroscience, February 23, 2011 • 31(8):2974 –2982
Behavioral/Systems/Cognitive
Olfactory Marker Protein Is Critical for Functional
Maturation of Olfactory Sensory Neurons and Development
of Mother Preference
Anderson C. Lee, Jiwei He, and Minghong Ma
Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Survival of many altricial animals critically depends on the sense of smell. Curiously, the olfactory system is rather immature at birth and
undergoes a maturation process, which is poorly understood. Using patch-clamp technique on mouse olfactory sensory neurons (OSNs)
with a defined odorant receptor, we demonstrate that OSNs exhibit functional maturation during the first month of postnatal life by
developing faster response kinetics, higher sensitivity, and most intriguingly, higher selectivity. OSNs expressing mouse odorant receptor
23 (MOR23) are relatively broadly tuned in neonates and become selective detectors for the cognate odorant within 2 weeks. Remarkably,
these changes are prevented by genetic ablation of olfactory marker protein (OMP), which is exclusively expressed in mature OSNs.
Biochemical and pharmacological evidence suggests that alteration in odorant-induced phosphorylation of signaling proteins underlie
some of the OMP⫺/⫺ phenotypes. Furthermore, in a novel behavioral assay in which the mouse pups are given a choice between the
biological mother and another unfamiliar lactating female, wild-type pups prefer the biological mother, while OMP knock-out pups fail
to show preference. These results reveal that OSNs undergo an OMP-dependent functional maturation process that coincides with early
development of the smell function, which is essential for pups to form preference for their mother.
Introduction
Many altricial animals give birth to close-eyed and relatively immobile pups, with early survival relying critically on the sense of
smell. When olfactory function is genetically disrupted in mice,
the pups die shortly after birth from inability to suckle (Brunet et
al., 1996; Belluscio et al., 1998; Wong et al., 2000). Curiously, the
olfactory system is relatively immature at birth and undergoes
significant development postnatally. Newborn rodents continue
to improve odor detection abilities (Alberts and May, 1980b) and
develop preference for familiar odors signaling mother, siblings,
home, and food (Schapiro and Salas, 1970; Gregory and Pfaff,
1971; Galef and Henderson, 1972; Alberts and Brunjes, 1978;
Brunjes and Alberts, 1979; Polan and Hofer, 1998). Associative
learning based on olfactory cues in early life effectively modifies
the brain circuits leading to behavioral attraction or avoidance
(Moriceau and Sullivan, 2006).
Odor detection relies on a large family of odorant receptors
(ORs) expressed in olfactory sensory neurons (OSNs) within the
Received Sept. 27, 2010; revised Dec. 13, 2010; accepted Dec. 27, 2010.
This work was supported by the National Institute on Deafness and Other Communication Disorders, National
Institutes of Health (R01 DC006213 to M.M. and fellowship to A.C.L.). We are grateful to Drs. Stephen Moss and Miho
Terunuma for guidance and equipment in obtaining the biochemical data. We thank Dr. Rui Feng and Yun Rose Li for
their advice and help with the statistical analysis, Kim Kridsada for her technical assistance in the behavioral tests,
and Drs. Huikai Tian and Tim Connelly for insightful discussions.
Correspondence should be addressed to Dr. Minghong Ma, Department of Neuroscience, University of
Pennsylvania School of Medicine, 215 Stemmler Hall, 3450 Hamilton Walk, Philadelphia, PA 19104. E-mail:
[email protected].
A. C. Lee’s present address: Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical
Institute, Columbia University, New York, NY 10032. E-mail: [email protected].
DOI:10.1523/JNEUROSCI.5067-10.2011
Copyright © 2011 the authors 0270-6474/11/312974-09$15.00/0
neuroepithelium of the nose. Binding of odor molecules to ORs
leads to increased intracellular cAMP levels via sequential activation of olfactory G-protein (Golf) and type III adenylyl cyclase
(ACIII). Subsequent opening of a cyclic nucleotide-gated (CNG)
channel and a Ca 2⫹-activated Cl ⫺ channel depolarizes OSNs
which then fire action potentials transmitting odor information
to the olfactory bulb in the brain (Su et al., 2009; Touhara and
Vosshall, 2009). OSNs from embryos or neonates are capable of
responding to odorants (Gesteland et al., 1982; Belluscio et al.,
1998). However, little is known about how these neurons mature
functionally during the postnatal life.
Olfactory marker protein (OMP) is widely recognized as a
molecular marker for mature OSNs (Margolis, 1972). Genetic
deletion of OMP results in slower odorant response kinetics revealed by electroolfactogram (EOG), single-cell recording, and
calcium imaging (Buiakova et al., 1996; Ivic et al., 2000; Reisert et
al., 2007; Kwon et al., 2009). Behaviorally, OMP⫺/⫺ mice exhibit
reduced odorant sensitivity and altered odor discrimination
(Youngentob and Margolis, 1999; Youngentob et al., 2001, 2004).
Because OMP expression is developmentally regulated, starting
at embryonic day 14 and reaching the adult level at postnatal day
30 (P30) (Graziadei et al., 1980), it is tempting to hypothesize that
OMP plays a role in functional maturation of OSNs.
Here we investigated whether OSNs expressing a defined OR
[mouse odorant receptor 23 (MOR23)] with a known ligand
(lyral) exhibit functional changes during the first postnatal
month. Using patch-clamp recordings, we demonstrated that
MOR23 neurons from P30 mice show faster response kinetics,
higher sensitivity, and higher selectivity than those from P0 mice.
Remarkably, OMP is required for this maturation process. Bio-
Lee et al. • OSN Maturation and Maternal Preference
chemical and pharmacological evidence supports our hypothesis
that altered ACIII phosphorylation contributes to the OMP⫺/⫺
phenotype. Furthermore, when pups are given a choice between
the biological mother and another unfamiliar lactating female,
wild-type but not OMP⫺/⫺ pups prefer the former to suckle or
huddle. These results reveal that OSNs undergo an OMPdependent functional maturation process that coincides with the
postnatal development of the smell function necessary for the
survival of altricial mice.
Materials and Methods
Animals. Genetically targeted MOR23-IRES (internal ribosome entry
site)-tauGFP mice were used to record MOR23 neurons that coexpress
green fluorescent protein (GFP) (Vassalli et al., 2002). To obtain
OMP⫺/⫺ MOR23 neurons, MOR23-IRES-tauGFP and OMP-synaptopHluorin (spH) mice (the coding region of OMP was replaced by that of
spH) (Bozza et al., 2004) were bred to generate homozygous double
mutant mice. All mice were in a mixed 129 and C57BL/6 genetic background and tested at ages from P0 to P30 as specified. P0 mice were
obtained by checking breeding pairs for new litters at 12 h intervals, and
the experiments were done within 12 h of detection to ensure recorded
cells were from pups within 24 h of birth. All procedures were approved
by the Institutional Animal Care and Use Committee at the University of
Pennsylvania.
Patch clamp. Intact epithelial preparations were as in our published
procedures (Ma et al., 1999; Grosmaitre et al., 2006). Mice were deeply
anesthetized either by intraperitoneal injection of ketamine/xylazine
(200 mg/kg and 15 mg/kg respectively) or 10 min incubation on ice (P0
and P7 pups), and then decapitated. The head was immediately put into
ice-cold Ringer’s solution, which contained (in mM): 124 NaCl, 3 KCl, 1.3
MgSO4, 2 CaCl2, 26 NaHCO3, 1.25 NaH2PO4, 15 glucose, pH 7.6, and
305 mOsm. The pH was kept at 7.4 after bubbling with 95% O2 and 5%
CO2. The nose was dissected out en bloc, the olfactory mucosa attached
to the nasal septum and the dorsal recess was removed and kept in oxygenated Ringer. Before use, the entire mucosa was peeled away from the
underlying bone and transferred to a recording chamber with the mucus
layer facing up. While recording, oxygenated Ringer was continuously
perfused at 25 ⫾ 2°C.
The dendritic knobs of OSNs were visualized through an upright microscope (Olympus BX61WI) with a 40⫻ water-immersion objective.
An extra 4⫻ magnification was achieved by an accessory lens in the light
path. The GFP-tagged cells were visualized under fluorescent illumination. Superimposition of the fluorescent and bright-field images allowed
identification of the fluorescent cells under bright field, which directed
the recording pipettes. Electrophysiological recordings were controlled
by an EPC-9 amplifier combined with Pulse software (HEKA). Perforated patch clamp was performed on the dendritic knobs by including
260 ␮M nystatin in the recording pipette, which was filled with the following solution (in mM): 70 KCl, 53 KOH, 30 methanesulfonic acid, 5.0
EGTA, 10 HEPES, 70 sucrose, pH 7.2 (KOH), and 310 mOsm. Under
voltage-clamp mode, the signals were initially filtered at 10 kHz and then
at 2.9 kHz. For odorant-induced transduction currents (which are slow
and long lasting), the signals were sampled at 333 Hz. Further filtering
offline at 60 Hz (to remove noise) did not change the response kinetics or
amplitudes, indicating that the sampling rate was sufficient and signal
aliasing was not a concern.
A seven-barrel pipette was used to deliver stimuli by pressure ejection
through a picospritzer (Pressure System IIe). The stimulus pipette was
placed ⬃20 ␮m downstream from the recording site, and both mechanical and chemical responses could be elicited in some neurons (Grosmaitre et al., 2007). All stimuli were delivered by 20 psi (138 kPa) via a
picospritzer. The pulse length was kept at 300 ms to ensure that the
neurons were stimulated by the intrapipette concentration (Grosmaitre
et al., 2006).
Odorants were prepared fresh daily in Ringer’s. Lyral was provided as
a generous gift from International Fragrances and Flavors, while (⫺)carvone, eugenol, heptanal, and geraniol were purchased from SigmaAldrich. Myristoylated autocamtide-2-related inhibitory peptide (AIP)
J. Neurosci., February 23, 2011 • 31(8):2974 –2982 • 2975
was purchased from Calbiochem and prepared before each experiment
from 1000⫻ DMSO stock. Okadaic acid (OA) was from Calbiochem and
prepared before each experiment from 100⫻ Ringer stock.
Behavioral tests. Three- to 6-month-old MOR23-IRES-tauGFP [wild
type (WT)] or OMP-spH/MOR23-IRES-tauGFP (OMP⫺/⫺) male and
female mice were paired to produce litters. Behavioral assays were conducted between 10:00 A.M. and 12:00 P.M. in an isolated room on a 7:00
A.M.–7:00 P.M. light cycle. For each assay, a clean mouse cage (8 ⫻ 12
inches) was used with cardboard barriers surrounding the cage and even
illumination to minimize environmental distractions. Two testing mothers
(called the stimulus animals) were anesthetized with ketamine/xylazine
(200 and 15 mg/kg, respectively) and placed 5–10 cm apart symmetrically
across the midline of the cage. For each assay, untrained test pups between the age of P7 and P16 were placed in between stimulus animals and
allowed to explore freely. Pups were tested individually or in groups (up
to 6 at a time), and both approaches produced similar results. Sometimes
the same pups were retested and similar results were always obtained as in
the initial test. Videotaping (Logitech QuickCam Pro 9000) was performed for 5–10 min until pups settled for ⬎3 min (most pups chose a
position to suckle or huddle within 2–5 min) and preference was judged
at the final position. The experiments were terminated and the data were
excluded if the animals did not stop exploring after 10 min or fell asleep
without exploring.
Isolation of ciliary membranes from OSNs. Ciliary membranes were
isolated using the high calcium method with minor modifications (Pace
et al., 1985; Sklar et al., 1986). The nasal epithelia from P30 mice were
surgically excised and pooled in 9 ml of solution C (in mM: 120 NaCl, 5
KCl, 1.6 K2HPO4, 1.2 MgSO4, 25 NaHCO3, 7.5 D-glucose, adjusted to pH
7.4). All steps were performed at 4°C if not otherwise stated. The pooled
tissue was allowed to settle to the bottom of the tube and the supernatant
was discarded. The tissue was resuspended in 5 ml of solution C containing 10 mM CaCl2 (solution D). Solution D was prepared by slowly adding
CaCl2 to a final concentration of 10 mM while gassing with 5% CO2/95%
air at 22°C to prevent Ca2PO4 from precipitation. The tissue was rocked
for 20 min (as in the mechanical agitation method) and then centrifuged
at 7700 ⫻ g for 5 min. This supernatant was reserved in a new tube for
subsequent pooling with membranes dislodged in a second rocking step.
The pellet was resuspended in 4 ml of solution D and rocked for a second
time for 20 min. Supernatant from this step, collected by centrifugation
at 7700 ⫻ g for 5 min, was added to the supernatant from the first calcium
shock incubation. The combined supernatant was centrifuged for 15 min
at higher speed (27 000 ⫻ g), supernatant was discarded, and resuspended in TEM buffer (10 mM Tris-HCl, 3 mM MgCl2, 2 mM EDTA, pH
8.0) and stored at ⫺70°C. Protein was determined by the Bradford Assay
(Bio-Rad), with bovine serum albumin as standard.
Immunoblot analysis. Membrane proteins were separated on 8% SDSpolyacrylamide gels and transferred to Hybond-C nitrocellulose membranes (GE Healthcare). Nonspecific binding sites were blocked with 5%
dry nonfat milk (Carnation) in TBST (10 mM Tris-HCl, pH 8.0, 150 mM
NaCl, and 0.4% Tween 20) for 1 h at room temperature. The blots were
incubated with primary antibody for 1 h at room temperature or overnight at 4°C. The primary antibodies used were rabbit anti-ACIII (1:
1000; Santa Cruz Biotechnology, sc-588), rabbit anti-Golf (1:1000; Santa
Cruz Biotechnology, sc-385), rabbit anti-phosphodiesterase 1C (PDE1C)
(1:1000; Abcam, ab14602), rabbit anti-PDE4A (1:1000; Abcam, ab14607),
mouse anti-␤-arrestin-2 (1:1000; Santa Cruz Biotechnology, sc-13140), and
mouse anti-␤-actin (1:5000; Sigma, A5441). Blots were washed three times
with TBST and then incubated with horseradish peroxidase (HRP)conjugated secondary antibody for 1 h at room temperature. The secondary
antibodies used were HRP-conjugated anti-rabbit antibody (1:5000; Millipore) and HRP-conjugated anti-mouse antibody (1:5000; Millipore). Blots
were washed twice with TBST and once with TBS (10 mM Tris-HCl, pH 8.0,
150 mM NaCl). ECL (Pierce) was used to visualize bound antibodies. Blots
were then quantified using the CCD-based FujiFilm LAS 3000 system.
Phosphorylation of ACIII. Purified cilia (4 ␮g) were resuspended with
50 ␮l of phosphorylation buffer containing (in mM): 50 HEPES-NaOH,
10 magnesium acetate, 0.5 CaCl2, pH 7.5. After incubation at 30°C for 1
min, cilia were treated with 10 ␮Ci [␥- 32P]ATP (specific activity, 3000
Ci/mmol; PerkinElmer) for another 1 min and then treated with a mix-
2976 • J. Neurosci., February 23, 2011 • 31(8):2974 –2982
Lee et al. • OSN Maturation and Maternal Preference
ture of 19 odorants each at 10 ␮M [heptanol, octanol, hexanal, heptanal,
octanal, heptanoic acid, octanoic acid, cineole, amyl acetate, (⫹)-limonene,
(⫺)-limonene, (⫹)-carvone, (⫺)-carvone, 2-heptanone, anisaldehyde,
benzaldehyde, acetophenone, 3-heptanone, and ethyl vanilline) for 1 min.
To terminate the reaction, 450 ␮l of cell lysis buffer (containing, in mM: 20
Tris-HCl, pH 8.0, 150 NaCl, 5 EDTA, 1% Triton X-100, 1 ␮g/ml leupeptin,
1 ␮g/ml aprotinin, and 0.5 phenylmethylsulfonyl fluoride) were added and
rotated for 1 h at 4°C. Lysates were then mixed with 2 ␮g of anti-ACIII
antibody (Santa Cruz Biotechnology) and protein A-Sepharose (20 ␮l of
50% slurry; GE Healthcare) and gently rotated for overnight at 4°C. The
beads were precipitated by centrifugation and washed twice with 1 ml of
cell lysis buffer, three times with 1 ml of washing buffer containing (in
mM): 20 Tris-HCl, pH 8.0, 500 NaCl, 5 EDTA, 1% Triton X-100, and
three times with 1 ml of cell lysis buffer. After the final wash, the beads
were resuspended in 50 ␮l of a sample buffer for SDS-PAGE, followed by
an electrophoresis, autoradiography, and analysis with a phosphor imager (Bio-Rad).
Statistical analysis. Three-way ANOVA tests, Bonferroni multiplecomparisons tests, logistic regression analysis, and ␹ 2 tests were performed using R Program. Dose–response curves were fit by GraphPad
Prism software. Student’s t tests were performed using a built-in function
in Excel (Microsoft). An average is shown as mean ⫾ SE unless otherwise
stated.
Results
OSNs exhibit faster response kinetics during development
To test whether OSNs undergo functional changes during development, we measured odorant responses in individual neurons
from animals at different ages (P0 –P30). We used a previously
characterized, gene-targeted mouse line (MOR23-IRES-tauGFP)
in which MOR23-expressing OSNs coexpress GFP (Vassalli et al.,
2002). For simplicity of description, these “green” cells are called
WT-MOR23 neurons in contrast to OMP⫺/⫺ MOR23 neurons.
Perforated patch-clamp recordings were performed on the dendritic knobs of individual OSNs in the intact epithelia (Grosmaitre et al., 2006).
First, we evaluated developmental changes in response kinetics and recorded a total of 73 WT-MOR23 neurons from P0, P7,
and P30 mice (n ⫽ 30, 21, and 22 cells, respectively). We quantified the following parameters: latency, rise time, peak current,
decay time, and residual current measured 10 s after the stimulation (Fig. 1 A). During the first postnatal month, the response
kinetics of WT-MOR23 neurons became significantly faster
(Figs. 1 B, 2). When stimulated by 10 ␮M lyral, the rise time
changed from 0.58 ⫾ 0.11 s (P0, n ⫽ 30) to 0.22 ⫾ 0.04 s (P30,
n ⫽ 22) or shortened by 62% (Fig. 1C), and the residual current
ratio (Iresidual/Ipeak) changed from 0.21 ⫾ 0.05 (P0) to 0.08 ⫾ 0.03
(P30), a 62% reduction (Fig. 1 D). Note that the response kinetics
depended on the odorant concentration within each age group
and similar trends were generally observed for the decay time and
the residual current ratio (Fig. 2). These results indicate that WTMOR23 neurons from P30 animals have both faster activation
and faster decay in odorant-elicited responses when compared
with their counterparts from neonates.
OMP deletion results in OSNs with slow response kinetics
To study the effects of OMP deletion on functional properties of
OSNs, we generated homozygous OMP⫺/⫺ mice with GFP labeled MOR23 neurons. Gene-targeted MOR23-IRES-tauGFP
and OMP-spH mice (Bozza et al., 2004) were bred to generate
homozygous double mutant mice. Note that the fluorescent spH
molecule is primarily expressed in the axon terminals of OSNs,
which allows unambiguous identification of GFP signals in the
cilia and dendritic knobs of MOR23 neurons in the olfactory
epithelium. We refer to these “green” cells as OMP⫺/⫺ MOR23
Figure 1. Wild-type but not OMP⫺/⫺ MOR23 neurons develop faster response kinetics
during the first month. A, Analysis of odorant-induced transduction currents under voltageclamp mode. The latency (1) is the time between the onset of the stimulus and the starting point
of the response. The rise time (2) is the time it takes for the current to reach 90% of the peak (3)
from the starting point of the response. The decay time (4) is the time it takes for the current to
return to 10% of the peak from the peak. Since some responses take minutes to return to the
baseline, a “residual” current (5) is measured 10 s after the stimulation. B, Inward currents
(normalized to the same peak) were elicited by lyral pulses from MOR23 neurons under different
conditions: WT P0 (thin blue), WT P30 (thick blue), OMP⫺/⫺ P0 (thin red) and OMP⫺/⫺ P30
(thick red). Representative traces from four single neurons were aligned at the start of the
responses. C, D, The rise time (C) and the ratio of the residual current to the peak (D) are
summarized for the four conditions: WT P0, WT P30, OMP⫺/⫺ P0, and OMP⫺/⫺ P30. The
holding potential was ⫺65 mV for all neurons. The output of the statistical analysis using
three-way ANOVA is detailed in Figure 2 D.
neurons. We recorded a total of 38 OMP⫺/⫺ MOR23 neurons: 11
from P0, 10 from P7, and 17 from P30 mice. Unlike WT-MOR23
neurons, OMP⫺/⫺ MOR23 neurons did not show significant
changes in response kinetics from P0 to P30 (Fig. 1 B). When
stimulated by 10 ␮M lyral, the rise time was 0.47 ⫾ 0.20 s (n ⫽ 11)
at P0 and 0.73 ⫾ 0.13 s (n ⫽ 17) at P30 (Fig. 1C), and the residual
current ratio (Iresidual/Ipeak) was 0.22 ⫾ 0.08 at P0 and 0.28 ⫾ 0.07
at P30 (Fig. 1 D). The lack of change in the response kinetics was
also observed with 1 and 100 ␮M lyral applications (Fig. 2 A–C).
Three-way ANOVA tests revealed that the activation phase (rise
time) depended on age and OMP status, while the decay phase
(decay time and Iresidual/Ipeak) depended on age, OMP status and
concentration (Fig. 2 D). The statistical analysis also confirmed
that lyral at higher concentrations induced responses with
shorter latency and higher peak current in all groups as expected
(Fig. 2 D). Generally, OMP⫺/⫺ MOR23 neurons at P30 responded to lyral similarly to WT-MOR23 neurons at P0, but with
significantly slower kinetics than their WT counterparts at P30,
suggesting that OMP is necessary for the kinetic changes during
development.
The signaling deficits of OMP deletion may be wide ranging,
but a recent study suggests that the action sites are upstream of
the CNG channel activation (Reisert et al., 2007). We examined
the protein levels of five key components (Golf, ACIII, PDE1C,
PDE4A and ␤-arrestin-2) in the signal transduction cascade and
found a significant upregulation of the ACIII level (Fig. 3 A, B).
Odor-induced phosphorylation, and subsequent inactivation of
Lee et al. • OSN Maturation and Maternal Preference
J. Neurosci., February 23, 2011 • 31(8):2974 –2982 • 2977
phorylation states may be one mechanism
underlying the OMP⫺/⫺ phenotype.
WT but not OMPⴚ/ⴚ OSNs exhibit
higher sensitivity during development
Next, we examined whether the sensitivity
of WT-MOR23 neurons changes during
the first postnatal month. WT-MOR23
neurons from P0 and P30 mice were stimulated by lyral at different concentrations
(0.01, 1, 10 and 100 ␮M) with an interval ⬎ 1 min to minimize odorant adaptation (Fig. 4 A, B). For each cell, the peak
current induced under each concentration is normalized to the maximum peak
current (typically induced by 100 ␮M
lyral). A single dose–response curve is
then fit based on the data from all neurons
within each age group, using a modified
Figure 2. The response kinetics of MOR23 neurons depends on age, OMP status, and concentration. A–C, Summary of the rise Hill equation, Inorm ⫽ (I ⫺ Ibaseline)/Imax ⫽
n
time (A), the decay time (B), and the ratio of the residual current to the peak (C) under different conditions: WT P0, WT P30, 1/(1 ⫹ (K1/2/C) , where I represents the
⫺/⫺
⫺/⫺
OMP
P0, and OMP
P30. All neurons were recorded under voltage-clamp mode with a holding potential of ⫺65 mV. Not peak current, Ibaseline the nonzero meshown are P7 data, which generally fall in between P0 and P30 data. D, Comparison of all five parameters is performed by chanical response induced by a Ringer
three-way ANOVA tests based on age (coded as an ordinal variable with levels 1, 2, and 3 corresponding to P0, P7, and P30, puff (Grosmaitre et al., 2007), Imax the
respectively), OMP status (coded as a categorical variable), and concentration (coded as an ordinal variable with level 1, 2 and 3 maximum current induced by the saturatcorresponding to 1, 10 and 100 ␮M, respectively). Significant p values are in bold. A significant p value in OMP status indicates ing concentration, K the concentration
1/2
difference between WT and OMP⫺/⫺ mice. A significant p value in age (or concentration) indicates that an increment in age (or
at which half of the maximum response
concentration) increases or decreases the parameter. Other significant differences not detailed in the main text include a longer
was reached, C the concentration of lyral,
latency of the responses in OMP⫺/⫺ neurons than in WT neurons and a decrease in peak currents with age.
and n the Hill coefficient. The mean K1/2
value shifted from 3.5 ␮M (n ⫽ 11) at P0 to
0.6 ␮M (n ⫽ 8) at P30 (Fig. 4 E, F ), indicating that WT-MOR23
ACIII by Ca 2⫹/calmodulin kinase II (CaMKII) contributes to
response termination (Wei et al., 1998). We reasoned that the
neurons become more sensitive during the first month. We then
long-lasting responses observed in OMP⫺/⫺ neurons could result
obtained the dose–response curves for OMP⫺/⫺ MOR23 neurons
from less ACIII phosphorylation, which leads to slower termina(Fig. 4C,D). The K1/2 value was 5.8 ␮M at P0 (n ⫽ 4) and 4.2 ␮M
at P30 (n ⫽ 10), not significantly different from each other, but
tion (Fig. 3C). We indeed observed a reduction in phosphorylasignificantly higher than the WT P30 value (Fig. 4 E, F ). Thus
tion of ACIII using a biochemical assay (Fig. 3D). We expected
OMP⫺/⫺ MOR23 neurons fail to increase their sensitivity by P30.
that blocking CaMKII (thus reducing ACIII phosphorylation)
would prolong the response decay in WT-MOR23 neurons,
WT but not OMPⴚ/ⴚ MOR23 neurons at P30 become
mimicking the OMP⫺/⫺ phenotype. When a membranepermeable CaMKII inhibitor, AIP (1 ␮M), was included in Ringselective lyral detectors
er’s solution, WT-MOR23 neurons at P30 exhibited slow decay
Last, we tested whether the selectivity of OSNs changes during the
(Iresidual/Ipeak ⫽ 0.21 ⫾ 0.07, n ⫽ 4) (Fig. 3 E, G, magenta). After
first postnatal month. In addition to lyral, we stimulated MOR23
1 h washout of AIP, WT-MOR23 neurons showed faster decay
neurons with four other odorants with distinct structural fea(Iresidual/Ipeak ⫽ 0.02 ⫾ 0.01, n ⫽ 4) (Fig. 3 E, G, green) with patures: (⫺)-carvone, eugenol, geraniol and heptanal at 100 ␮M if
rameters similar to the untreated controls (Fig. 1 B, thick blue).
not otherwise stated (Fig. 5). Only cells that were tested by the
Conversely, we expected that blocking phosphatase activity
complete set of five odorants plus Ringer are included in the
(Boekhoff and Breer, 1992; Kroner et al., 1996), thus increasing
following analysis. Confirming earlier identification of lyral as
ACIII phosphorylation, would shorten the response decay of
the cognate ligand of MOR23 (Touhara et al., 1999), all WTOMP⫺/⫺ MOR23 neurons, reversing the OMP⫺/⫺ phenotype.
MOR23 neurons at P30 (n ⫽ 9) responded to lyral (Fig. 5C), and
When a phosphatase inhibitor, OA (at 1 ␮M), was included in
only two cells displayed responses to a nonlyral odorant with
Ringer’s solution, OMP⫺/⫺ MOR23 neurons at P30 exhibited
peak amplitude ⬎ 10% of the lyral response. Remarkably, WTfaster decay (Iresidual/Ipeak ⫽ 0.02 ⫾ 0.02, n ⫽ 5) (Fig. 3 F, G,
MOR23 cells (n ⫽ 9) from P0 mice responded to many odorants
green), similar to WT-MOR23 neurons at P30 (Fig. 1 B, thick
and were not specifically tuned to lyral (Fig. 5A). Nine of nine
blue). After 1 h washout of OA, OMP⫺/⫺ MOR23 neurons
cells (100%) responded to a nonspecific ligand with peak amplishowed slow decay (Iresidual/Ipeak ⫽ 0.28 ⫾ 0.06, n ⫽ 4) (Fig.
tude ⬎ 10% of the lyral response, with five of these cells respond3 F, G, green), similar to untreated OMP⫺/⫺ MOR23 neurons at
ing to a nonspecific ligand greater than the lyral response. We
P30 (Fig. 1 B, thick red). The results reveal that reducing phosthen tested WT-MOR23 cells from P9 –P15 mice (age matched to
phorylation by blocking CaMKII is sufficient to slow the response
the behavioral tests described below), which were similar to their
decay, while increasing phosphorylation by blocking phosphacounterparts from P30 mice (Fig. 5B). These data reveal that
tase activity is sufficient to hasten the response decay. Although
WT-MOR23 neurons, which are relatively nonselective at birth,
we cannot rule out the possibility that AIP and OA exert their
undergo a maturation process in their ligand selectivity and beactions on targets other than ACIII, these results support our
come selective lyral detectors postnatally. We then tested whether
hypothesis. Modification of ACIII (and/or other proteins) phosOMP is necessary for the selectivity change observed in WT-
Lee et al. • OSN Maturation and Maternal Preference
2978 • J. Neurosci., February 23, 2011 • 31(8):2974 –2982
MOR23 neurons during development. Similar to their WT counterparts, all nine OMP⫺/⫺ MOR23 neurons from P0 mice
responded to multiple odorants (Fig. 5D) and 89% (8 of 9) of the
cells responded to a nonspecific ligand with peak amplitude ⬎
10% of the lyral response. Strikingly, most OMP⫺/⫺ MOR23 neurons from P9 –P15 or from P30 mice continued to respond to
many nonspecific odorants instead of being specifically tuned to
lyral (Fig. 5 E, F ). Sixty-seven percent (12 of 18) and 88% (14 of
16) of the cells from P9 –P15 and P30 OMP⫺/⫺ mice, respectively,
responded to another ligand with peak amplitude ⬎ 10% of lyral
response. All three OMP⫺/⫺ groups contained some cells responding to a nonspecific ligand greater than the lyral response
(Fig. 5D–F ). Using logistic regression analysis, we compared the
percentage of cells responding to a nonlyral odorant with peak
amplitude ⬎ 10% of the lyral response in different groups. The
analysis showed that age and OMP status are two critical factors
in determining the tuning property of MOR23 neurons. P0
groups are more likely to be broadly responsive compared with
P30 groups (odds ratio ⫽ 21.4, p ⬍ 0.05), whereas P9 –P15
groups are similar to P30 groups. OMP⫺/⫺ groups are more likely
to be broadly responsive than the WT groups (odds ratio ⫽ 12.4,
p ⬍ 0.001). These data indicate that OMP⫺/⫺ MOR23 neurons
fail to become specific lyral detectors by P30.
Figure 3. Deletion of OMP alters ACIII signaling. A, The signaling proteins from the olfactory
epithelia obtained from WT and OMP⫺/⫺ P30 mice were stained by specific antibodies in
Western blots. Three of the five tested proteins are shown here. Note that ACIII protein has a
smear-like band according to the manufacture’s datasheet. B, The expression levels of three
signaling proteins in OMP⫺/⫺ mice are normalized to those in WT animals. The ACIII level in the
olfactory epithelium was ⬃75% higher in OMP⫺/⫺ mice (n ⫽ 5 samples) with adjusted p ⬍
0.05 (*) in t test with Bonferroni correction. C, Schematic drawing illustrates that phosphorylation of ACIII by CaMKII inhibits its enzyme activity, while dephosphorylation by phosphatase
resets its activity. Phosphorylation and dephosphorylation are represented by green and magenta, respectively. D, OMP deletion reduced odorant-induced ACIII phosphorylation. ACIII
phosphorylation was measured by incubating purified olfactory cilia with (⫹) or without (⫺)
an odorant mixture with [␥- 32P]ATP for 1 min. ACIII protein was immunoprecipitated and
resolved with SDS-PAGE, and radiolabeled phospho-ACIII was visualized on x-ray film. Basal
ACIII phosphorylation was ⬃10% higher in OMP⫺/⫺ mice. Odorant-induced phosphorylation
of ACIII increased by ⬃2.3-fold in WT animals, and only ⬃1.5 in OMP⫺/⫺ animals. Similar
results were obtained from two samples. Considering that ACIII protein is upregulated in
WT but not OMPⴚ/ⴚ pups prefer the biological mother
Since OSNs undergo functional maturation during the first
month of life, we expected to observe some behavioral deficits in
the development of smell function. We devised a maternal preference assay to test the ability of pups to discriminate their biological mother from another lactating but unfamiliar female. The
two mothers were anesthetized and placed 5–10 cm apart in the
center of a clean cage (Fig. 6 A, B). The test pups aged P7–P16
were placed between the stimulus animals and allowed to explore
the cage freely. Pups younger than P7 were generally not active
enough, while those older than P16 often did not stop exploring
the new environment within 10 min. With both mothers anesthetized, vocalization, retrieval and temperature influences are
eliminated, and the behavior of pups is driven primarily by olfactory cues. Most pups attempted to suckle within 2–5 min, and
preference was determined by the position of the pup nose relative to the midline after they settled (Fig. 6 A, B). To ensure that
the WT and OMP⫺/⫺ pups were tested under similar conditions,
one WT and one OMP⫺/⫺ mother were paired in most of the
experiments and their age matched pups were tested consecutively. Most of the WT pups (78%, n ⫽ 45 pups from 7 litters)
preferred to suckle or huddle with their biological mother over
the unfamiliar lactating female (Fig. 6 A, C). Remarkably,
OMP⫺/⫺ pups failed to show maternal preference, with 55% (n ⫽
58 from 9 litters) suckling or huddling with their biological
mother (Fig. 6 B, C). To rule out that this finding was due to some
deficits of the OMP⫺/⫺ mothers, we also performed the same test
4
OMP⫺/⫺ mice (A, B), the reduction in phosphorylated ACIII in OMP⫺/⫺ mice is even more
significant. E, Block of ACIII phosphorylation via inhibition of CaMKII slows odorant response
decay in WT-MOR23 neurons from P30 mice. Inward currents (normalized to the same peak)
were elicited by brief lyral (10 ␮M) pulses in the presence of 1 ␮M AIP (magenta) and after
washout (green). F, Block of ACIII dephosphorylation via inhibiting phosphatase activity hastens odor response decay in OMP⫺/⫺ MOR23 neurons from P30 mice. Inward currents (normalized to the same peak) were elicited by brief lyral (10 ␮M) pulses in the presence of 1 ␮M OA
(green) and after washout (magenta). The holding potential was ⫺65 mV for all neurons. G,
TheratiooftheresidualcurrenttothepeakissummarizedforthefourconditionsinEandF.Unpaired
Student’s t test (assuming equal variances and two-tail) is used: *p ⬍ 0.05 and **p ⬍ 0.01.
Lee et al. • OSN Maturation and Maternal Preference
J. Neurosci., February 23, 2011 • 31(8):2974 –2982 • 2979
dents (Welker, 1964; Alberts and May,
1980a) and may allow OSNs to faithfully
detect odorants at a higher frequency. In
addition, OSNs exhibit higher sensitivity
during postnatal development (Fig. 4).
The dynamic range of MOR23 neurons in
responding to lyral covers 3– 4 orders of
magnitude in all age groups, but the averaged K1/2 value changes from 3.5 ␮M at P0
to 0.6 ␮M at P30, equivalent to an ⬃6-fold
increase in sensitivity. In this study, we
recorded odorant-induced inward currents in the dendritic knobs of individual
OSNs, which represent a relatively direct
measurement of the transduction events.
It would be interesting to know how these
transduction currents convert into action
potentials which carry the odor information to the brain. We did not compare
odorant-induced firing patterns in this
study because we only observed action
potentials in some but not all MOR23
neurons upon odorant stimulation under
current-clamp mode. It is possible that
⫺/⫺
Figure 4. Wild-type but not OMP
MOR23 neurons increase the sensitivity to lyral during the first month. A–D, Inward some action potentials are generated relacurrents were induced by lyral at various concentrations (0.01–100 ␮M) under different conditions: WT P0 (A), WT P30 (B), tively far out in the axon, and that they do
OMP⫺/⫺ P0 (C), and OMP⫺/⫺ P30 (D). The holding potential was ⫺65 mV for all neurons. The traces were from four individual not backpropagate into the soma and
neurons. E, The dose–response curves are plotted for different conditions. F, The K1/2 value is summarized for different conditions. dendrites. The increased sensitivity of the
We performed Bonferroni multiple comparisons for logK1/2 among the four groups. MOR23 has significantly lower logK1/2 com- primary sensory neurons potentially conpared with the other three groups (***adjusted p ⬍ 0.001 for all 3 comparisons), whereas there is no difference among the other tributes to the improved smell ability at
three. For easier comprehension, the mean K1/2 ⫾ 95% confidence interval (CI) is plotted here.
the behavioral level during postnatal development (Alberts and May, 1980b).
The most striking change of OSNs durusing two WT mothers. Similar preference was obtained in the
ing the first postnatal month is that they exhibit increased selecWT pups (81% or 21 of 26 from four litters). Additional control
tivity (Fig. 5). MOR23 neurons from P9 –P15 and P30 mice are
experiments revealed that mouse pups (WT or OMP⫺/⫺) prerelatively selective to lyral, consistent with previous ligand idenferred lactating females over nonlactating females (both are unfatification among a panel of diverse odorants (Touhara et al.,
miliar; Fig. 6C), confirming that OMP⫺/⫺ pups did not have
1999). Surprisingly, MOR23 neurons from P0 mice are broadly
deficits in finding lactating nipples or suckling. These results
responsive to distinct odorants, often with amplitudes comparasuggest that OMP-dependent maturation of OSNs is necessary
ble or even larger than lyral responses. This phenomenon is unfor mouse pups to develop preference for the biological
likely restricted to the MOR23 receptor, because randomly
mother.
recorded rodent OSNs become more selective during early develDiscussion
opment (Gesteland et al., 1982). At present, we can only speculate
Here we report that mouse OSNs undergo a functional maturathe underlying mechanisms. One possibility is that young OSNs
tion process by showing faster response kinetics, higher sensitivstart with multiple ORs and gradually one OR becomes dominant
ity, and higher selectivity from P0 to P30. Genetic ablation of OMP
by suppressing the expression of other ORs. However, coexpresprevents the response property changes during postnatal developsion of multiple ORs or gene switching in OSNs is infrequently
ment, indicating a critical role of OMP in functional maturation of
encountered even at early stages (Shykind et al., 2004; Tian and
OSNs. Altered odorant-induced phosphorylation of ACIII may proMa, 2008). Another possibility is that OSNs from newborn mice
vide a molecular and cellular mechanism underlying the slow decay
use different signal transduction cascades compared with those
observed in OMP⫺/⫺ neurons. In a mother preference assay, WT
from P30 mice. This is supported by the fact that newborn Golfpups prefer their biological mother over another unfamiliar lactating
null mice exhibit odorant-induced EOG signals, probably via sigfemale, while OMP⫺/⫺ pups fail to show preference. The current
naling of other G-proteins (Belluscio et al., 1998; Lin et al., 2007).
study reveals OMP-dependent functional maturation of OSNs that
Other potential mechanisms underlying the selectivity difference
coincides with the postnatal development of the smell function.
between P0 and P30 include differential gap junction connecTo examine functional alterations of OSNs during postnatal
tions, mucus components (e.g., odorant binding proteins), and
development, we compared the odorant responses of MOR23
modification of odorant receptor proteins.
neurons from mice at different ages (P0 to P30). Our data indeed
OMP has been widely used as a marker for mature OSNs since
reveal significant changes in the following three aspects, which
its initial identification (Margolis, 1972), but its functions are still
potentially improve the smell function. OSNs exhibit faster actielusive. Here we demonstrated that OSNs undergo a functional
vation and decay kinetics in responding to odorants during postmaturation process and OMP is required in this process. Deletion
natal development (Figs. 1, 2). Interestingly, such changes
of OMP in OSNs from P30 animals leads to response properties
coincide with the postnatal increment of the sniffing rate in roresembling those from newborns in all three aspects tested. First,
2980 • J. Neurosci., February 23, 2011 • 31(8):2974 –2982
Lee et al. • OSN Maturation and Maternal Preference
Figure 5. Wild-type but not OMP⫺/⫺ MOR23 neurons increase the selectivity during the first month. A–F, Odorant-induced transduction currents were recorded in MOR23 neurons under
different conditions: WT P0 (A), WT P9 –P15 (B), WT P30 (C), OMP⫺/⫺ P0 (D), OMP⫺/⫺ P9 –P15 (E) and OMP⫺/⫺ P30 (F). Each scatter plot in the bottom row shows the peak currents induced by
different stimuli in individual cells under the corresponding condition. Each color represents a single cell. When a peak current exceeds 200 pA, it is plotted at 200 pA for the purpose of visualization.
All odorants were delivered at 100 ␮M (except two cells in WT P0 tested by 10 ␮M) and the holding potential was ⫺65 mV for all neurons.
Figure 6. Wild-type but not OMP⫺/⫺ pups prefer the biological mother. A, B, WT pups preferred to suckle or huddle with the biological mother over another unfamiliar lactating female (A), while
OMP⫺/⫺ mice failed to show preference (B). The nose of each pup is marked by a dot and its location relative to the midline (dashed line) between the two anesthetized mothers is used to assign
the pup’s preference. C, Summary of mother preference for WT and OMP⫺/⫺ pups. The numbers in parentheses indicate the total number of pups tested in each group. Statistical analysis was
performed using a ␹ 2 test. The choice between a lactating and a nonlactating female (both are unfamiliar) was tested from two litters of each genotype.
the response kinetics (both activation and decay phase) is
slower in OMP⫺/⫺ neurons (Figs. 1, 2), which is consistent
with previous EOG and single-cell recordings (Buiakova et al.,
1996; Ivic et al., 2000; Reisert et al., 2007). This agreement
suggests that the OMP-null phenotype in the current study
should not be restricted to MOR23 neurons but generally applicable to OSNs expressing other ORs. Second, the response
sensitivity is significantly reduced in OMP⫺/⫺ OSNs. At P30,
the sensitivity of MOR23 neurons in responding to lyral is
⬃7-fold higher with OMP (K1/2 ⫽ 0.6 ␮M) compared with that
without OMP (K1/2 ⫽ 4.2 ␮M) (Fig. 4). The reduced sensitivity
of OMP⫺/⫺ OSNs may contribute to the higher detection
threshold found in OMP-null mice in behavioral tests (Youngentob and Margolis, 1999), which can be rescued by viral transfection of OMP (Youngentob et al., 2004). Third, OMP⫺/⫺ MOR23
neurons from P30 mice fail to become specific lyral detectors
(Fig. 5). These data suggest that OMP⫺/⫺ OSNs are functionally
“locked” in an immature stage in odorant response properties.
However, this does not imply that these properties are achieved
by the same cellular mechanisms.
With the wide-range of deficits in OMP⫺/⫺ OSNs, it is plausible that OMP exerts its molecular and cellular actions at multi-
Lee et al. • OSN Maturation and Maternal Preference
ple sites. Our biochemical and pharmacological experiments
support that ACIII is one of the proteins that is modulated by
OMP deletion. Although the total ACIII level is upregulated in
OMP⫺/⫺ mice, odorant-induced phosphorylation of ACIII is reduced (Fig. 3), which potentially leads to slower termination of the
responses (Wei et al., 1998). Indeed, blocking phosphorylation via a
CaMKII inhibitor in WT OSNs mimics the slow termination in
OMP⫺/⫺ phenotype. Conversely, enhancing phosphorylation by
blocking phosphatase activity restores the fast termination in
OMP⫺/⫺ OSNs (Fig. 3). Phosphatase 2A is likely involved in this
process, because it is especially sensitive to okadaic acid (Kroner
et al., 1996). Although the results of the pharmacological experiments are consistent with our hypothesis on modulation of
ACIII in OMP⫺/⫺ phenotype, there may be other proteins (such
as odorant receptors) serving as substrates for phosphorylation
and dephosphorylation (Boekhoff and Breer, 1992). Nevertheless, our results revealed a critical role of CaMKII and phosphatase 2A in determining the response kinetics in OSNs. OMP
deletion also causes a trend of upregulation of PDE4A and
␤-arrestin-2 ( p ⫽ 0.07 for both proteins in t test for Western
blots expression levels), confirmed by immunostaining in the
olfactory epithelium. PDE4A potentially helps to remove the intracellular cAMP (Cygnar and Zhao, 2009) and ␤-arrestin-2 is
involved in OR desensitization (Mashukova et al., 2006). Further
studies are needed to dissect out the contributions of each of these
signaling proteins in OMP phenotype.
We used a simple mother preference test to investigate the
potential impact of maturation of OSNs on early development of
smell function in the animal. We found that WT pups (P7 to P16)
preferred the biological mother to suckle or huddle with over an
unfamiliar lactating female. In contrast, OMP⫺/⫺ pups did not
show preference, revealing a novel behavioral deficit in OMP
knock-out mice (Fig. 6). This behavior is most likely mediated by
olfactory cues because some of the pups did not open their eyes
when the tests were performed. With both mothers anesthetized,
it is unlikely the pups used auditory, thermal, or somatosensory
cues to identify their biological mother.
Different odor-guided behaviors appear to function at different developmental stages and may be influenced by OMP deletion to different degrees. For instance, neonates rely on the
olfactory cues to initiate suckling (Blass and Teicher, 1980) and
OMP⫺/⫺ pups are normal in this aspect. During the early postnatal period, rodent pups gradually develop preference for huddling with conspecifics (Alberts and Brunjes, 1978; Brunjes and
Alberts, 1979), for home odors (Gregory and Pfaff, 1971), and for
mother odors (Polan and Hofer, 1998). Here we demonstrated
that WT mouse pups prefer their own mother over an unfamiliar
lactating female (Fig. 6). A common feature of these behaviors is
that rodent pups are attracted to familiar odors, which could
result from genetic relatedness (Todrank et al., 2005) and/or
experience-dependent learning (Wilson and Sullivan, 1994).
Genetic ablation of OMP leads to mouse pups that do not
show preference for their biological mother, which likely results
from disruption of the functional maturation process of OSNs.
The results of the behavioral tests should extend beyond the special case of MOR23 mice. Curiously, the mere capability of OSNs
in responding to odors is not sufficient for the pups to smell
properly. For instance, odorant-induced EOG signals can be elicited from Golf knock-out mice at P0, but these pups die from
starvation shortly after birth. Our results on the selectivity of
OSNs at P0 may have provided an explanation. When a large
fraction of OSNs responds to many odorants in a nonselective
manner, the animals have impaired capability to discriminate the
J. Neurosci., February 23, 2011 • 31(8):2974 –2982 • 2981
odorants (Fleischmann et al., 2008). Similarly, OMP⫺/⫺ OSNs
can respond to odorants but with less selectivity (Fig. 5), which
may be one of the factors leading to improper development of
mother preference observed here.
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